Project Summary Numerous mutations associated with various forms of hereditary hearing loss (HHL) have been identified in many genes. Defining the physiological roles of HHL genes and the pathological mechanisms of mutations found in these genes is important for the development of remedies against HHL. This study will determine the pathogenicity and pathological mechanisms of mutations found in two HHL genes, SLC26A4 and SLC26A5. SLC26A4 encodes an anion transporter, pendrin. Alterations of this gene are one of the common causes of hereditary hearing loss, and over 300 missense mutations have been identified in this gene. In order to define the pathological mechanisms of these mutations, it is important to know how these mutations affect the function of pendrin and how the functional phenotypes correlate with disease phenotypes. To this end, protein expression, membrane targeting, and anion transport functions of mutated pendrin proteins will be characterized in a mammalian cell line. The results will be mapped onto a structural model so that mechanistic insights for pendrin and other SLC26 proteins can be obtained. The large number of missense mutations found in SLC26A4 makes these efforts systematic and thorough. SLC26A5 encodes a membrane-based motor protein, prestin, which is abundantly expressed in OHCs. Its voltage-driven motor activity, electromotility, is demonstrated to be essential for normal cochlear amplification. However, it remains unclear how electromotility is used in the amplification process. Pathological characterizations of SLC26A5 mutations have been ambiguous due to the lack of this fundamental knowledge. In this study, the importance of the rapid motor activity of prestin will be examined using an animal model expressing prestin harboring an HHL-associated mutation that significantly slows the motor kinetics of prestin. In addition, two novel SLC26A5 missense mutations recently found in a patient with congenital hearing loss will be characterized in order to further gain insights as to how prestin contributes to the cochlear amplification process and OHC maintenance. Functional characterization of disease-associated mutations is not only important for assessing their pathogenicity, but also beneficial for appreciating the normal physiological roles of genes and defining the molecular mechanisms of the gene products. Successful completion of this study allows unequivocal pathological characterization of mutations found in SLC26A4 and SLC26A5.!